Magnetic separation of biological entities from fluid sample

ABSTRACT

The present disclosure relates to, inter alia, devices, systems, and methods for use in the magnetic separation of biological entities from fluid samples. This device includes a magnetic separation chamber configured to receive a fluid sample for magnetic separation, where the magnetic separation chamber includes at least two magnets mounted on the surface or in the wall of the magnetic separation chamber. The device also includes a force provider configured to move the magnetic separation chamber in a side-to-side motion to mix and/or magnetize the fluid sample. In one embodiment, the magnetic separation chamber is in a form of a sleeve and comprises a substantially central channel for loading a vessel containing the fluid sample therein. The systems and methods of the present disclosure involve the use of this device to separate biological entities from fluid samples.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Phase filing under 35 U.S.C. § 371of International Application No. PCT/US2019/042887, filed Jul. 22, 2019,and published as WO 2020/019001 A1 on Jan. 23, 2020, which claimspriority benefit of U.S. Provisional Patent Application Ser. No.62/701,557, filed Jul. 20, 2018, the disclosures of which are herebyincorporated by reference herein in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under Grant Number1343058 awarded by the National Science Foundation. The Government hascertain rights in the invention.

FIELD OF THE INVENTION

The present disclosure relates to, inter alia, devices, systems, andmethods relating to the magnetic separation of biological entities fromfluid samples.

BACKGROUND OF THE INVENTION

The US blood testing market is estimated at $20 billion and is expectedto increase to $30 billion by 2030 (1). This predicted growth stems froman increasing demand for CLIA-waived testing environments, such asUrgent Care and Minute Clinics, that offer diagnostic capabilities atlower costs and greater convenience than conventional laboratorytesting. CLIA-waived clinics embody a shift in the healthcare landscapetowards decentralization and higher accessibility of medical testing.The separation of unwanted cellular material is critical for theaccuracy and reliability of many molecular diagnostics tools (2), forexample many blood tests require that plasma is separated from red bloodcells prior to analysis. In commercial blood testing laboratories,centrifuges are almost exclusively employed to perform separation and isa key first step to facilitate accurate quantitative diagnostics (3).However, the US Food and Drug Administration (FDA) classifiescentrifuges as ‘moderately’ complex devices that are unsuitable for usein CLIA-waived environments (4). This constraint has bottlenecked thetranslational ability of diagnostic technologies as centrifuges areunable to adapt for use at the point-of-need and are becomingincreasingly obsolete in a landscape that is seeking the furtherdecentralization of testing services.

Passive filtration membranes are a solution to perform cell separationat the point-of-need and are often used with lateral flow assays (5).Enabled by capillary action, the membranes operate on the principle ofselectively preventing particles of a certain size from flowing throughthem. While inexpensive and easy to manufacture, the performance ofthese membranes is marred by inconsistencies in separation caused byclogged pores. Inconsistencies manifest themselves as variability inamount of plasma that is obtained during each run which deters theperformance of quantitative lateral flow tests. Immunochemistry issensitive to a variation in amount of sample that is used for analysis(6,7). Additionally, the recovery rates of these methods are low (8)(less than 30% of available plasma) which presents a barrier foranalysis of analytes at low concentrations and sizes (9).

Several microfluidic approaches have been demonstrated in literaturethat achieve high levels of separation. These methods can be broadlyclassified into “active” and “passive” techniques (10). Activetechniques employ an external field (acoustic, electric or magnetic)that is used to align or immobilize the blood cells so as to enable theplasma to be separated in a continuous flow format. Passive methodstypically separate the cells using hydrodynamic effects or separatingpillars using cleverly designed and intricate microfluidic fabricationarchitecture (11-13). While these techniques have been excellentdemonstrative proof-of-concepts, they lack the ability to becommercialized as microfluidic fabrication is a highly involved andexpensive process that lacks scalability (14). Further, in the case ofactive methods, the complex designs are often too cost-ineffective tointegrate into existing microfluidic methods rendering them impracticalfor use at the point-of-need (9). To address the high cost and lack ofaccessibility, many researchers have used common household items andtoys like salad spinners and egg beaters to achieve plasma separation(15-17). These designs were cleverly engineered to simulatecentrifugation but are not robust enough to operate in point-of-needclinical settings. There is a need for low-cost technologies that canoffer highly efficient blood-plasma separation at the point-of-need withhigh reliability and efficiency. A ‘simple’ method would further enablepresent day diagnostics to transition for use at the point-of-need(18-21) and enable the decentralization of blood testing services.

The present invention is directed to overcoming these and otherdeficiencies in the art.

SUMMARY OF THE INVENTION

The present disclosure relates to, inter alia, devices, systems, andmethods for use in the magnetic separation of biological entities fromfluid samples.

In one aspect, the present disclosure provides a device for use inmagnetic separation of a target biological entity from a fluid sample.The device includes: (a) a magnetic separation chamber configured toreceive a fluid sample for magnetic separation, wherein said magneticseparation chamber comprises two magnets mounted on opposing sidewalls asufficient distance from one another so as to prevent interferencebetween the magnetic field of each magnet, and wherein the magneticseparation chamber is configured to receive and maintain the fluidsample at a position between the two magnets during operation of thedevice; and (b) an actuator functionally coupled to the magneticseparation chamber, wherein said actuator comprises a linear actuatorconfigured to move the magnetic separation chamber laterally in aside-to-side motion so as to keep the two magnets in line with the fluidsample during operation of the actuator.

In another aspect, the present disclosure provides a system for use inmagnetic separation of a target biological entity from a fluid sample.This system includes: (a) at least one device according to the presentdisclosure; and (b) a plurality of magnetic beads each functionalized tobind to the target biological entity, thereby being effective to captureand separate the target biological entity from the fluid sample.

In another aspect, the present disclosure provides a method forseparating a target biological entity from a fluid sample. This methodinvolves the steps of: (a), said method comprising the steps of: (a)providing a device according to the present disclosure; (b) combiningthe fluid sample with a plurality of magnetic beads functionalized tobind to the target biological entity, wherein the combining is performedin a vessel comprising a tube or other suitable container for containingthe fluid sample during operation of the device; (c) positioning thevessel containing the fluid sample and the functionalized magnetic beadsbetween the two magnets of the magnetic separation chamber of thedevice; and (d) operating the device in a manner sufficient to capturethe target biological entity on the functionalized magnetic beads,thereby yielding a supernatant free of or substantially free of saidtarget biological entity.

In another aspect, the present disclosure is directed to a device foruse in magnetic separation of a target biological entity from a fluidsample. This device includes: (a) a magnetic separation chamberconfigured to receive a fluid sample for magnetic separation, whereinsaid magnetic separation chamber comprises at least two magnets mountedon the surface or in the wall of the magnetic separation chamber; and(b) a force provider configured to move the magnetic separation chamberin a side-to-side motion to mix and/or magnetize the fluid sample. Inone embodiment, the magnetic separation chamber is in a form of a sleeveand comprises a substantially central channel for loading a vesselcontaining the fluid sample therein, and further includes at least twoside channels each containing at least one magnet therein. In certainembodiments, the side channels are positioned at opposite ends andopposite sides of the substantially central channel.

In another aspect, the present disclosure is directed to a method forseparating a target biological entity from a fluid sample. The methodinvolves the steps of: (a) providing a device according to the presentdisclosure; (b) combining the fluid sample with a plurality of magneticbeads functionalized to bind to the target biological entity, whereinthe combining is performed in a vessel comprising a tube or othersuitable container for containing the fluid sample during operation ofthe device; (c) positioning the vessel containing the fluid sample andthe functionalized magnetic beads between the two magnets of themagnetic separation chamber of the device; and (d) operating the devicein a manner sufficient to capture the target biological entity on thefunctionalized magnetic beads, thereby yielding a supernatant free of orsubstantially free of said target biological entity. In one embodiment,the magnetic separation chamber is in a form of a sleeve and comprises asubstantially central channel for loading a vessel containing the fluidsample therein, and further includes at least two side channels eachcontaining at least one magnet therein. In certain embodiments, the sidechannels are positioned at opposite ends and opposite sides of thesubstantially central channel.

These and other objects, features, and advantages of this invention willbecome apparent from the following detailed description of the variousaspects of the invention taken in conjunction with the accompanyingdrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of illustrating aspects of the present invention, thereare depicted in the drawings certain embodiments of the invention.However, the invention is not limited to the precise arrangements andinstrumentalities of the embodiments depicted in the drawings. Further,if provided, like reference numerals contained in the drawings are meantto identify similar or identical elements.

FIGS. 1A-1D are schematic drawings of different view of one embodimentof a device of the present disclosure. FIG. 1A is an exploded view ofthe device. FIG. 1B is a perspective view of the device. FIG. 1C is atop view of the device. FIG. 1D is a side view of the device.

FIG. 2 is a schematic of one embodiment of a system of the presentdisclosure, which also illustrates a method of using the system.

FIG. 3 is a flow chart of one embodiment of a method of magneticseparation of biological entities from a fluid sample in accordance withthe present disclosure.

FIGS. 4A-4D are schematic drawings of embodiments of the device, system,and method of the present disclosure. FIG. 4A illustrates the use of theportable benchtop H.E.R.M.E.S device and system in accordance with amethod of the present disclosure. As shown in FIG. 4A, in oneembodiment, the method involves three simple steps: (i) collect and loadthe sample in a test tube precoated with functionalized magnetic beads,(ii) place sample in H.E.R.M.E.S and wait for 90 seconds, and (iii)remove sample and extract plasma using a capillary tube. FIG. 4Billustrates a size comparison of the portable benchtop H.E.R.M.E.Sdevice against a standard laboratory centrifuge (Fisherbrand accuSpinMicro 17). FIG. 4C illustrates a side and top profile view of a portablebenchtop device of the present disclosure. FIG. 4D illustrates anexploded view of a portable benchtop H.E.R.M.E.S device of the presentdisclosure, showing its internal components.

FIGS. 5A-5B illustrate how a target biological entity (red blood cells)are aggregated and captured using an aggregation agent andfunctionalized magnetic beads. FIG. 5A illustrates the effect ofaggregation of blood cells prior to capture. FIG. 5B illustratesseparation of plasma from captured red blood cells with application of amagnetic field in accordance with the present disclosure, as seen undera microscope.

FIG. 6 is a graph illustrating separation of plasma from human sampleswith artificially high hematocrit values. Each sample was tested induplicate and the standard deviation for each sample set is indicatedwith error bars (n=2). An average purity of 99.9% was obtained in thesesamples.

FIGS. 7A-7B illustrate the effect of the aggregation agent onperformance of the H.E.R.M.E.S. methodology. FIG. 7A is a graph of aplot showing the effect of the aggregation agent on performance ofH.E.R.M.E.S. Three different sample volumes were used as noted in thelegend. A non-linear relationship is observed between the amount ofblood cells and the amount of aggregation agent required to capturethem. An average of 34 μL and 53 μL of total recovered plasma wereobtained in the cases of samples tested using 80 and 120 μL volumes.Standard deviation for each sample is indicated with error bars. (n=2).The inset shows that the concentration of magnetic beads does not affectthe performance significantly. FIG. 7B are images of separated plasma atthree different aggregation agent concentrations. From this figure it isclear that a concentration of around 3.5 μg/mL is optimal to obtainhighly pure plasma.

FIGS. 8A-8C illustrate the performance of various aspects of theH.E.R.M.E.S methodology. FIG. 8A is a graph of a plot comparing theperformance of plasma obtained from H.E.R.M.E.S and commerciallyavailable filtration paper. A starting volume of 40 μL was used for eachtest and 15 μL of plasma was used for the strips that were used withH.E.R.M.E.S. The pictures on the left and right side of the plot areimages of test strips run using plasma obtained from H.E.R.M.E.S andfiltration paper respectively. In general, one can note a higher T/Cratio for all the samples that used H.E.R.M.E.S for separation. Further,one can observe a higher coefficient of determination R² and a sharperslope in the case of samples processed with H.E.R.M.E.S. The standarddeviation of each sample (n=2) is indicated by error bars. FIG. 8B is agraph illustrating a comparison of the mean coefficient of variations(CV=standard deviation/mean*100) between the two types of plasma. The CVof all samples from H.E.R.M.E.S was lower than 10%. FIG. 8C is a graphillustrating a comparison of the ROC curves (generated using the Delongmethod). The a.u.c from samples purified by H.E.R.M.E.S was 1 and thea.u.c from filtered samples was 0.789.

FIGS. 9A-9B are schematic drawings of one embodiment of a magneticseparation chamber in the form of a sleeve for use in magneticseparation of a biological entity from a fluid sample according to thepresent disclosure. FIG. 9A illustrates an external view of the sleeve.FIG. 9B illustrates an internal cross-sectional view of the sleeve.

FIG. 10 is a schematic drawing illustrating one embodiment of how thesleeve device illustrated in FIGS. 9A-9B can be used for the magneticseparation of a biological entity from a fluid sample according to thepresent disclosure.

DESCRIPTION OF THE INVENTION

Disclosed herein are, inter alia, devices, systems, and methods relatingto the magnetic separation of biological entities from fluid samples.

Device for Use in Magnetic Separation of a Target Biological Entity froma Fluid Sample

In one aspect, the present disclosure is directed to a device for use inmagnetic separation of a target biological entity from a fluid sample.The device includes: (a) a magnetic separation chamber configured toreceive a fluid sample for magnetic separation, wherein said magneticseparation chamber comprises two magnets mounted on opposing sidewalls asufficient distance from one another so as to prevent interferencebetween the magnetic field of each magnet, and wherein the magneticseparation chamber is configured to receive and maintain the fluidsample at a position between the two magnets during operation of thedevice; and (b) an actuator functionally coupled to the magneticseparation chamber, wherein said actuator comprises a linear actuatorconfigured to move the magnetic separation chamber laterally in aside-to-side motion so as to keep the two magnets in line with the fluidsample during operation of the actuator.

In one embodiment, the magnetic separation chamber is configured toreceive a fluid sample having a volume of up to 200 microliters.

In one embodiment, the magnetic separation chamber is configured toreceive a vessel comprising a tube or other suitable containercontaining the fluid sample.

In one embodiment, the magnets are circular magnets.

In one embodiment, the magnets are neodymium magnets.

In one embodiment, the actuator is an actuating solenoid. A suitableactuating solenoid for use in the present disclosure can include,without limitation, a linear actuator solenoid.

In one embodiment, the actuating solenoid is a 12 volt actuatingsolenoid.

In one embodiment, the device further includes a housing unit configuredto house the magnetic separation chamber and the actuator.

In one embodiment, the housing unit comprises a base portion and a topcover portion, wherein the base portion is configured to hold themagnetic separation chamber and the actuator, and wherein the top coverportion is configured to fit over and cover the base portion and itscontents.

In one embodiment, the top cover portion further comprises an openingfor inserting the fluid sample into a position between the two magnetsof the magnetic separation chamber.

In one embodiment, the housing unit is configured to further houseonboard electronics components effective to operate the actuator and toenable automation of the device.

In one embodiment, the onboard electronics components comprise at leastone component selected from the group consisting of a microcontroller, atransistor, and a power input jack.

In one embodiment, the power input jack is suitable for use with astandard 12 volt, 0.5 ampere wall power supply. In other embodiments,the device of the present disclosure can be battery powered.

In one embodiment, the device is in a form of a portable benchtopdevice.

The device according to claim 14, wherein the portable benchtop devicehas dimensions not greater than 4 centimeters in height, 5 centimetersin width, and 8 centimeters in length.

In one embodiment, the device is in a form suitable for use forpoint-of-need diagnostics of the fluid sample.

Turning to FIGS. 1A-1D, there is illustrated one embodiment of device 10of the present disclosure. As shown, device 10 can be used for magneticseparation of a target biological entity from a fluid sample. In certainembodiments, device 10 includes magnetic separation chamber 20configured to receive a fluid sample for magnetic separation. As shownin FIG. 1A, magnetic separation chamber 20 includes two magnets (22 a,22 b) mounted on opposing sidewalls (24 a, 24 b) a sufficient distancefrom one another so as to prevent interference between the magneticfield of each magnet (22 a, 22 b). Magnetic separation chamber 20 isconfigured to receive and maintain the fluid sample at a positionbetween the two magnets (22 a, 22 b) during operation of device 10.Device 10 also includes actuator 30 functionally coupled to magneticseparation chamber 20, where actuator 30 includes linear actuator 32configured to move the magnetic separation chamber 20 laterally in aside-to-side motion so as to keep the two magnets (22 a, 22 b) in linewith the fluid sample during operation of actuator 30.

As shown in FIGS. 1A-1D, in certain embodiments, device 10 furtherincludes housing unit 50 configured to house magnetic separation chamber20 and actuator 30. In certain embodiments, housing unit 50 includesbase portion 52 and top cover portion 54. Base portion 52 can beconfigured to hold magnetic separation chamber 20 and actuator 30, wheretop cover portion 54 is configured to fit over and cover base portion 52and its contents (e.g., magnetic separation chamber 20, actuator 30 withlinear actuator 32, microcontroller 62, transistor 64, and power inputjack 66). As shown, top cover portion 54 can further include opening 56for inserting the fluid sample into a position between the two magnets(22 a, 22 b) of magnetic separation chamber 20. In one embodiment,housing unit 50 is configured to further house onboard electronicscomponents 60 effective to operate actuator 30 and to enable automationof device 10. In one embodiment, onboard electronics components 60 caninclude, without limitation, at least one component selected frommicrocontroller 62, transistor 64, and/or power input jack 66. As shownin FIG. 1D, in certain embodiments, power input jack 66 is suitable foruse with a standard 12 volt, 0.5 ampere wall power supply 70.

As shown in FIG. 1B, in one embodiment, device 10 is in a form of aportable benchtop device. Without intending to limit the scope of thedescribed device, as illustrated in FIG. 1B, in a particular embodiment,portable benchtop device 10 can have dimensions not greater than 4centimeters in height, 5 centimeters in width, and 8 centimeters inlength. Such dimensions are particularly suitable for use forpoint-of-need diagnostics of fluid samples.

System for Use in Magnetic Separation of a Target Biological Entity froma Fluid Sample

In another aspect, the present disclosure is directed to a system foruse in magnetic separation of a target biological entity from a fluidsample. This system includes: (a) at least one device according to thepresent disclosure; and (b) a plurality of magnetic beads eachfunctionalized to bind to the target biological entity, thereby beingeffective to capture and separate the target biological entity from thefluid sample.

In one embodiment, the system further includes an aggregation agentconfigured to group together an aggregate of a plurality of the targetbiological entities prior to the capture and separation of the targetbiological entity from the fluid sample.

In one embodiment, the aggregation agent is selected from the groupconsisting of an antibody, a protein, dextran, polyvinylpyrollidone,polyoxyethylene, fibrinogen, immunoglobulin IgM and IgG, calciumchloride, and combinations thereof.

In one embodiment, the target biological entity are red blood cells andthe aggregation agent is an antibody that binds to red blood cellsnon-specifically, irrespective of blood type.

In one embodiment, the aggregation agent is an antibody that binds to asurface marker of red blood cells.

In one embodiment, the surface marker of the red blood cell is selectedfrom the group consisting of CD235a and TER119.

In one embodiment, the plurality of magnetic beads and the aggregationagent are contained in a vessel comprising a tube or other suitablecontainer for containing the fluid sample during operation of thedevice.

In one embodiment, the plurality of magnetic beads and the aggregationagent are provided together in a storage buffer.

In one embodiment, the plurality of magnetic beads are conjugated to theaggregation agent prior to adding the fluid sample.

In one embodiment, the plurality of magnetic beads and the aggregationagent are dried directly in a collection container prior to adding thefluid sample.

In one embodiment, the collection container is a vessel comprising atube or other suitable container for containing the fluid sample duringoperation of the device.

In one embodiment, the target biological entity is selected from thegroup consisting of white blood cells, contaminants, waste products, andexcess reagents contained in the fluid sample.

In one embodiment, the system further includes a collection componentfor collecting a supernatant produced after capture and separation ofthe target biological entity from the fluid sample.

In one embodiment, the collection component is a capillary tubeconfigured to passively uptake the supernatant.

In one embodiment, the fluid sample is a blood sample, the targetbiological entity comprises red blood cells, and the supernatantcomprises plasma.

Turning to FIG. 2, there is illustrated one embodiment of how device 10and system 105 can be used in a method of magnetic separation of targetbiological entities 84 from fluid sample 80 in accordance with thepresent disclosure. As shown in FIG. 2, in one embodiment, system 105includes at least one device 10 according to the present disclosure anda plurality of magnetic beads 82 each functionalized to bind to targetbiological entity 84, thereby being effective to capture and separatetarget biological entity 84 from fluid sample 80. As shown in FIG. 2, inone embodiment, the plurality of magnetic beads 82 and aggregation agent86 are contained in vessel 90, where vessel 90 can be a tube or othersuitable container for containing fluid sample 80 during operation ofdevice 10. In one embodiment, system 105 further includes collectioncomponent 92 for collecting a supernatant produced after capture andseparation of target biological entity 84 from fluid sample 80. As shownin FIG. 2, in one embodiment, collection component 92 is a capillarytube configured to passively uptake the supernatant.

Method for Use in Magnetic Separation of a Target Biological Entity froma Fluid Sample

In another aspect, the present disclosure is directed to a method forseparating a target biological entity from a fluid sample. This methodinvolves the steps of: (a) providing a device according to the presentdisclosure; (b) combining the fluid sample with a plurality of magneticbeads functionalized to bind to the target biological entity, whereinthe combining is performed in a vessel comprising a tube or othersuitable container for containing the fluid sample during operation ofthe device; (c) positioning the vessel containing the fluid sample andthe functionalized magnetic beads between the two magnets of themagnetic separation chamber of the device; and (d) operating the devicein a manner sufficient to capture the target biological entity on thefunctionalized magnetic beads, thereby yielding a supernatant free of orsubstantially free of said target biological entity.

In one embodiment, the operating step comprises running the actuator toenable the magnetic separation chamber to function to capture the targetbiological entity with the functionalized magnetic beads and concentratethem proximate to just one of the two magnets, thereby separating thetarget biological entity from the fluid sample.

In one embodiment, running the actuator comprises moving the magneticseparation chamber laterally in a side-to-side motion with respect tothe fluid sample for a sufficient number of strokes to homogenouslydistribute the functionalized magnetic beads among the target biologicalentity and concentrate them proximate to just one of the two magnets,thereby separating the target biological entity from the fluid sample.

In one embodiment, the number of strokes is not greater than 100strokes, not greater than 50 strokes, not greater than 40 strokes, notgreater than 30 strokes, not greater than 20 strokes, or not greaterthan 10 strokes.

In one embodiment, the sufficient number of strokes is completed withinnot more than 180 seconds, not more than 150 seconds, not more than 120seconds, not more than 90 seconds, not more than 60 seconds, or not morethan 30 seconds.

In one embodiment, the method further involves collecting thesupernatant from the vessel.

In one embodiment, the collecting is performed using a capillary tube.

In one embodiment, the method further involves collecting and analyzingthe supernatant and/or the captured target biological entity using oneor more diagnostic tool or technique of interest. Suitable diagnostictools and techniques of interest are well known in the art andcontemplated for use with the devices, systems, and methods of thepresent disclosure.

In one embodiment, the method does not involve a centrifugation step toseparate the target biological entity from the fluid sample.

In one embodiment, the magnetic beads are conjugated to an aggregationagent prior to the combining step, and wherein said aggregation agent isconfigured to group together an aggregate of a plurality of the targetbiological entities prior to the capture and separation of the targetbiological entity from the fluid sample.

In one embodiment, the aggregation agent is selected from the groupconsisting of an antibody, a protein, dextran, polyvinylpyrollidone,polyoxyethylene, fibrinogen, immunoglobulin IgM and IgG, calciumchloride, and combinations thereof.

In one embodiment, the target biological entity are red blood cells andthe aggregation agent is an antibody that binds to red blood cellsnon-specifically, irrespective of blood type.

In one embodiment, the aggregation agent is an antibody that binds to asurface marker of red blood cells.

In one embodiment, the surface marker of the red blood cell is selectedfrom the group consisting of CD235a and TER119.

In one embodiment, the plurality of magnetic beads and the aggregationagent are contained in a vessel comprising a tube or other suitablecontainer for containing the fluid sample during operation of thedevice.

In one embodiment, the plurality of magnetic beads and the aggregationagent are provided together in a storage buffer.

In one embodiment, the plurality of magnetic beads are conjugated to theaggregation agent prior to adding the fluid sample.

In one embodiment, the plurality of magnetic beads and the aggregationagent are dried directly in a collection container prior to adding thefluid sample.

In one embodiment, the collection container is a vessel comprising atube or other suitable container for containing the fluid sample duringoperation of the device.

In one embodiment, the target biological entity is selected from thegroup consisting of white blood cells, contaminants, waste products, andexcess reagents contained in the fluid sample.

In one embodiment, the method further involves using a collectioncomponent for collecting a supernatant produced after capture andseparation of the target biological entity from the fluid sample.

In one embodiment, the collection component is a capillary tubeconfigured to passively uptake the supernatant.

In one embodiment, the fluid sample is a blood sample, the targetbiological entity comprises red blood cells, and the supernatantcomprises plasma.

Turning to FIG. 3, there is illustrated one embodiment of a method forseparating a target biological entity from a fluid sample. As shown inthe flow chart of FIG. 3, separation method 100 involves: (a) step 110:providing a device according to the present disclosure; (b) step 120:combining the fluid sample with a plurality of magnetic beadsfunctionalized to bind to the target biological entity, wherein thecombining is performed in a vessel comprising a tube or other suitablecontainer for containing the fluid sample during operation of thedevice; (c) step 130: positioning the vessel containing the fluid sampleand the functionalized magnetic beads between the two magnets of themagnetic separation chamber of the device; and (d) step 140: operatingthe device in a manner sufficient to capture the target biologicalentity on the functionalized magnetic beads, thereby yielding asupernatant free of or substantially free of said target biologicalentity. In one embodiment, method 100 further involves step 150:collecting the supernatant from the vessel. In one embodiment, method100 further involves step 160: collecting and analyzing the supernatantand/or the captured target biological entity using one or morediagnostic tool or technique of interest.

Further Attributes of the Disclosed Device, System, and Method ofMagnetic Separation

As provided herein, in various aspects, the devices, systems, andmethods of magnetic separation of as various attributes andcharacteristics over the existing art. In a particular embodiment, theaggregation agent is effective to aggregate the biological entity withinthe sample and the magnetic separation means is effective to mix,capture, and isolate the aggregated biological entity from the sample.

In one embodiment, the magnetic separation means can include a pair ofmagnets configured to generate a mixing effect within the sample.

In one embodiment, the system can further include magnetic beadsconjugated to the aggregation agent.

In one embodiment, the magnetic separation means can include a pair ofmagnets configured to homogenously distribute the magneticbeads/aggregation agent among the biological entity.

In one embodiment, the sample is blood. However, the sample can be anybiological sample from any biological source.

In one embodiment, the biological entity can include, withoutlimitation, red blood cells, white blood cells, contaminants, wasteproducts, excess reagents, and the like. However, the biological entitycan include any entity of interest that is contained in the sample.

In one embodiment, the system used in the method includes an aggregationagent and a magnetic separation means.

In one embodiment, the aggregation agent used in the method is effectiveto aggregate the biological entity within the sample and the magneticseparation means is effective to mix, capture, and isolate theaggregated biological entity from the sample.

In one embodiment, the magnetic separation means used in the method caninclude, without limitation, a pair of magnets configured to generate amixing effect within the sample.

In one embodiment, magnetic beads are conjugated to the aggregationagent used in the method.

In one embodiment, the magnetic separation means used in the method caninclude, without limitation, a pair of magnets configured tohomogenously distribute the magnetic beads/aggregation agent among thebiological entity.

In one embodiment, the system used in the method can further include acapillary collection component, with the capillary collection componentbeing used to collect the isolated biological entity. In a particularembodiment, the capillary collection component is a capillary tube.

In one aspect the present disclosure involves the use of functionalizedmagnetic beads in combination with a stable aggregation agent to captureand separate biological entities of interest (e.g., blood cells, fatcells) from a fluid sample using an external magnetic field. Thepurified sample can be extracted using a capillary tube.

In one aspect, the present disclosure provides a system referred toherein as High Efficiency Rapid Magnetic Erythrocyte Separator(abbreviated herein as H.E.R.M.E.S or HERMES), a portable low-costsystem that enables the separation and extraction of red blood cellsfrom plasma within a short period of time (e.g., within 2 minutes).Broadly speaking, HERMES can perform highly efficient separation ofbiological agents from a biological sample. With the aggregation agent,this system and associated method are particularly useful for capturingbiological entities that are present in large concentrations withrespect to other entities in the sample. For example, the system andmethod can be used in situations that require the removal ofcontaminants, waste products or excess reagents in a biological sample.

Provided below are illustrative examples of various components, aspects,and embodiments of the HERMES system and associated methods.

Aggregation agent—In certain embodiments, HERMES uses a stableaggregation agent to clump red blood cells together thereby reducing thenumber of effective targets that need to be captured. Calculations showthat a 2000-fold increase in efficiency is noticed when the aggregationagent is added. Note that the blood cells have been used just as anexample. The aggregation agent can be used for any biological agent ofinterest.

In one embodiment, the aggregation agent can be an antibody that bindsto red blood cells non-specifically. As understood in the art,antibodies for blood cells generally refer to antibodies specific toantigens present on the surface of the red blood cell. Thus, whendetermining the blood type of a person, i.e., type A, type B, type AB,and type 0, references to A and B are specific antigens while type 0simply refers to “having no antigen.”

In one embodiment, the aggregation agent can be an antibody that bindsnon-specifically to every red blood cell irrespective of blood type (onesource being Rockland antibodies). Thus, it is not necessary to knowwhat the antibody binds to on the surface of the cell. In certainembodiments, the antibody can bind to a marker known as CD235a which isknown to be expressed on the surface of all mature red blood cells.There are other known cell markers like TER119 and so on. In aparticular embodiment, the aggregation agent is a protein that iscapable of non-specifically binding to the surface markers of red bloodcells (or the entity that needs to be captured). Other suitableaggregation agents can include, without limitation, dextran,polyvinylpyrollidone, polyoxyethylene, fibrinogen, immunoglobulin IgMand IgG, calcium chloride, and the like.

Contact free mixing—In certain embodiments, HERMES uses a pair ofmagnets that are moved in a particular direction with respect to thesample in order to homogenously distribute the beads amongst the bloodcells. Efficient mixing enables highly efficient capture. This alsoremoves the need for other lab equipment like pipetting devices, vortexmixers etc. In a particular case, a linear actuator is used tomanipulate the sample in one direction hereby simplifying the captureprocess as we can use the same magnetic field for capture. For example,the two magnetic fields are placed far enough away from each other so asto not cause an interference with each other.

Dry stored reagents—In certain embodiments, the beads and aggregationagent are dried directly in the tube so there is no dilution that takesplace due to addition of liquid reagents. This means that the resultingplasma can be used directly in an immunoassay or similar form ofdiagnostic test without the need for additional processing. Note thatplasma is used in this specific example but whatever resulting fluidremains from separation can be appropriately substituted in this claimas well.

No washing step—In certain embodiments, since the objective is only inobtaining erythrocyte depleted plasma there is no need for a washingstep to get rid of excess reagents.

Capillary collection—In certain embodiments, once the plasma isseparated it is collected using a capillary tube that can passivelyuptake the plasma once it is dipped. This feeds in to the previous claimof not needing lab equipment such as pipettes.

Single Vessel—In certain embodiments, HERMES uses a single vessel toperform sample collection, capture, separation and extraction. Thesample is not moved from the vessel until extraction.

Other Applications for HERMES:

Various embodiments of the HERMES system and associated methods can beused in a wide variety of applications involving the separation ofbiological agents from a biological sample. Below are some specificillustrative examples of other applications of the HERMES system andmethods.

White blood cell isolation—In certain embodiments, HERMES can be used asa technology that isolates white blood cells instead of a technologythat removes red blood cells. White blood cells contain DNA that can beused for genetic testing. In some cases, they can also contain DNA ofinfectious agents. In this situation, one could use Polymerase ChainReaction methods to amplify the DNA and analyze it.

Hematocrit Determination—In certain embodiments, once the red bloodcells are isolated using HERMES, one can use image capture and analysisto quantify the amount of red blood cells present in the sample therebygiving a value for hematocrit, which is a useful check for anemia or acomplete blood count.

Aspects of one embodiment of the present invention are described in moredetail in Example 1, which details the design and principle of the HighEfficiency Rapid Magnetic Erythrocyte Separator (H.E.R.M.E.S), aportable low-cost system that enables the separation and extraction ofred blood cells from plasma within 2 minutes. H.E.R.M.E.S usesfunctionalized magnetic beads to capture and separate red blood cellsand achieves near perfect separation, rivaling the efficiency of that ofa commercial centrifuge while using inexpensive raw materials.H.E.R.M.E.S employs a standalone protocol that does not require the useof any specialized lab equipment such as pipettes. As shown in Example1, the efficacy of H.E.R.M.E.S is demonstrated with the help of humansamples, and further proves that H.E.R.M.E.S improves the performance ofexisting lateral flow assays in comparison to commercially availablefiltration membranes.

Sleeve Device and Use Thereof in Magnetic Separation

As used herein, the term “HERMES sleeve” is meant to broadly cover anymagnetic separation chamber of the present disclosure that is in theform of a sleeve (see e.g., FIGS. 9A, 9B, and 10). Therefore, as used inthis context, the term “HERMES” is not meant to limit the HERMES sleeveto the use of magnetic separation of “erythrocytes” from the fluidsamples, but is meant to cover the use of the sleeve in the magneticseparation of any target biological entity from any type of fluidsample.

In one aspect, the present disclosure is directed to a device for use inmagnetic separation of a target biological entity from a fluid sample.This device includes: (a) a magnetic separation chamber configured toreceive a fluid sample for magnetic separation, wherein said magneticseparation chamber comprises at least two magnets mounted on the surfaceor in the wall of the magnetic separation chamber; and (b) a forceprovider configured to move the magnetic separation chamber in aside-to-side motion to mix and/or magnetize the fluid sample. In oneembodiment, the magnetic separation chamber is in a form of a sleeve andcomprises a substantially central channel for loading a vesselcontaining the fluid sample therein.

In one embodiment, the force provider is an actuator or a movable hand.

In one embodiment, the force provider is an actuator and the two magnetsare mounted on opposing sidewalls a sufficient distance from one anotherso as to prevent interference between the magnetic field of each magnet,wherein the magnetic separation chamber is configured to receive andmaintain the fluid sample at a position between the two magnets duringoperation of the device, and wherein the actuator is functionallycoupled to the magnetic separation chamber, said actuator comprising alinear actuator configured to move the magnetic separation chamberlaterally in a side-to-side motion so as to keep the two magnets in linewith the fluid sample during operation of the actuator.

In one embodiment, the at least two magnets are fixed or movable alongthe wall of the magnetic separation chamber.

In one embodiment, the magnetic separation chamber further comprises atleast two channels along the sidewall of the chamber to allow the atleast two magnets movable in order to mix and magnetize the sample.

In one embodiment, the at least two channels are linear channels, curvedchannels, symmetric channels, cylindrical shaped, or tube shaped.

In one embodiment, the force provider is a manual force provider or anautomatic force provider.

In one embodiment, the fluid sample further comprises at least anaggregation agent configured to group together an aggregate of aplurality of the target biological entities.

In one embodiment, the device further comprises a collection componentfor collecting a supernatant produced after capture and separation ofthe target biological entity from the fluid sample, and wherein thecollection component is cap comprising capillary tube.

In one embodiment, the magnetic separation chamber comprising a durableplastic material or any other non-magnetic material.

In one embodiment, the at least two magnets are ball magnets.

In one embodiment, the ball magnets are 3/16 inch ball magnets (KJMagnetics).

In one embodiment, the magnetic separation chamber is in a form of asleeve and comprises a substantially central channel for loading avessel containing the fluid sample therein.

In one embodiment, the substantially central channel has a circumferencesuitable for a standard blood collection tube.

In one embodiment, the substantially central channel has a circumferenceof approximately 10 mm and a depth of approximately 70-75 mm.

In one embodiment, the sleeve is configured to contain a vesselcomprising a 3 mL tube.

In one embodiment, the vessel can contain a volume of fluid sampleselected from the group consisting of up to 200 uL, up to 500 uL, up to1 mL, up to 1.5 mL, up to 2.0 mL, up to 2.5 mL, up to 3.0 mL, and up to5 mL.

In one embodiment, the vessels can contain a volume of fluid sample of200 uL or greater.

In one embodiment, the sleeve is made with 3D printed plastic.

In one embodiment, the sleeve is made with injection molding techniques.

In one embodiment, the sleeve comprises dimensions approximating thoseof a 10 mL blood tube, said dimensions comprising approximately 16 mm incircumference and 100 mm in length for the depth of the substantiallycentral channel, wherein total size of the sleeve can alternativelyscale up or down according to these dimensions accordingly.

In one embodiment, the size of the sleeve can be scaled up at least byapproximately 1.5 times.

In another aspect, the present disclosure is directed to a method forseparating a target biological entity from a fluid sample. The methodinvolves the steps of: (a) providing a device according to the presentdisclosure; (b) combining the fluid sample with a plurality of magneticbeads functionalized to bind to the target biological entity, whereinthe combining is performed in a vessel comprising a tube or othersuitable container for containing the fluid sample during operation ofthe device; (c) positioning the vessel containing the fluid sample andthe functionalized magnetic beads between the two magnets of themagnetic separation chamber of the device; and (d) operating the devicein a manner sufficient to capture the target biological entity on thefunctionalized magnetic beads, thereby yielding a supernatant free of orsubstantially free of said target biological entity.

In one embodiment this method further involves collecting thesupernatant from the vessel.

In one embodiment this method further involves collecting and analyzingthe supernatant and/or the captured target biological entity using oneor more diagnostic tool or technique of interest.

In one embodiment, the method does not involve a centrifugation step toseparate the target biological entity from the fluid sample.

In one embodiment, the magnetic beads are conjugated to an aggregationagent prior to the combining step, and wherein said aggregation agent isconfigured to group together an aggregate of a plurality of the targetbiological entities prior to the capture and separation of the targetbiological entity from the fluid sample.

In one embodiment, the aggregation agent is selected from the groupconsisting of an antibody, a protein, dextran, polyvinylpyrollidone,polyoxyethylene, fibrinogen, immunoglobulin IgM and IgG, calciumchloride, and combinations thereof.

In one embodiment, the target biological entity are red blood cells andthe aggregation agent is an antibody that binds to red blood cellsnon-specifically, irrespective of blood type.

In one embodiment, the aggregation agent is an antibody that binds to asurface marker of red blood cells.

In one embodiment, the surface marker of the red blood cell is selectedfrom the group consisting of CD235a and TER119.

In one embodiment, the plurality of magnetic beads and the aggregationagent are contained in a vessel comprising a tube or other suitablecontainer for containing the fluid sample during operation of thedevice.

In one embodiment, the plurality of magnetic beads and the aggregationagent are provided together in a storage buffer.

In one embodiment, the plurality of magnetic beads are conjugated to theaggregation agent prior to adding the fluid sample.

In one embodiment, the plurality of magnetic beads and the aggregationagent are dried directly in a collection container prior to adding thefluid sample.

In one embodiment, the collection container is a vessel comprising atube or other suitable container for containing the fluid sample duringoperation of the device.

In one embodiment, the target biological entity is selected from thegroup consisting of white blood cells, contaminants, waste products, andexcess reagents contained in the fluid sample.

In one embodiment, this method further involves a collection componentfor collecting a supernatant produced after capture and separation ofthe target biological entity from the fluid sample.

In one embodiment, the collection component is a capillary tubeconfigured to passively uptake the supernatant.

In one embodiment, the fluid sample is a blood sample, the targetbiological entity comprises red blood cells, and the supernatantcomprises plasma.

Turning to FIGS. 9A-9B and 10, there is illustrated device 200 in theform of a sleeve for use in magnetic separation of a biological entityfrom a fluid sample according to the present disclosure. FIG. 9Aillustrates an external view of sleeve device 200. FIG. 9B illustratesan internal cross-sectional view of sleeve device 200. As shown, sleevedevice 200 includes magnetic separation chamber 214 having top portion210 and bottom portion 212. As shown, bottom portion 212 can be curved,but need not be. Top portion 210 includes opening 500 of substantiallycentral channel 510. Substantially central channel 510 is where vessel600 (containing the fluid sample) is housed during operation of sleevedevice 200. As shown in FIG. 9B, sleeve device 200 includessubstantially central channel 510 and two side channels (300 a, 300 b)that each contain magnets (400 a, 400 b).

FIGS. 9A and 9B and position 1 of FIG. 10 show sleeve device 200oriented in an upright position, where top portion 210 is facing up andbottom portion 212 is facing down. As shown in FIG. 10, in this uprightposition (position 1, FIG. 10), magnet 400 a of side channel 300 a is afarther distance away from substantially central channel 510 as comparedto magnet 400 b of side channel 300 b. Further, in this orientation, thebulk of the fluid sample containing the target biological entity willcollect in vessel 600 toward the bottom of vessel 600 (also the bottomof substantially central channel 510). As depicted in FIG. 10 (position1), this also results in magnet 400 b coming into contact to the rightside of substantially central channel 510. Therefore, as shown in FIG.10, during operation, when sleeve device 200 is moved to an upside downposition (position 2), where top portion 210 is facing down and bottomportion 212 is facing up, vessel 600 will be inverted with the bulk ofthe fluid sample being at the top of vessel 600. Also in this upsidedown position (position 2, FIG. 10), magnet 400 a contained in sidechannel 300 a will be a closer distance to the substantially centralchannel 510 compared to magnet 400 b of side channel 300 b. As depictedin FIG. 10 (position 2), this also results in magnet 400 a of sidechannel 300 a coming into contact to the left side of substantiallycentral channel 510. Continuous inversions of sleeve device 200 from theupright position to the upside down position results in lateral mixingof the contents of the fluid sample due to the side-to-side magneticfield changes, as well as gravitational mixing of the fluid in alongitudinal manner within vessel 600.

In certain embodiments, the HERMES sleeve offers portable separationcapabilities for blood for sample volumes of up to 1.5 mL, withoutlimitation. The HERMES sleeve capitalizes on the innovation of theoriginal portable benchtop HERMES device and system as described herein.The sleeve mimics the lateral mixing of the benchtop HERMES device whiletaking advantage of bulk-scale mixing that is generally created byend-over-end mixing. In certain embodiments, this sleeve may be moreappropriate for use in the field and other translational settings.

In certain embodiments, the benefit of this sleeve is at least two-fold:(a) the sleeve is resource independent and fully self-sufficient; and(b) the sleeve is compatible with macroscale volumes (up to 1.5 mL).

In certain examples, the HERMES sleeve has been tested with 7 humansamples. All human samples were tested with 1.5 mL of blood. Theexperiments were sufficient to attain an average purity of 99.9% with ayield of 67%. In one example, the sleeve was designed much like acommercially available tube holder. In this example, the sleeve wascylindrical in shape and includes a hole in the center to mount astandard blood collection tube. Inside the sleeve, there were 2 chambersthat each housed a high strength spherical ball magnet (e.g. 3/16 inchball magnet purchases from KJ Magnetics). As seen in FIG. 10, theconfiguration of the magnets with respect to the sample changes as thetube is inverted.

During operation of certain examples of the HERMES sleeve, once thesample is collected in the tube it can be placed in the HERMES sleevefor mixing. Mixing is facilitated by inversion. As shown FIG. 10, inposition 1, the beads are magnetized on the left-hand side of the tube,while in position 2 (inverted), the beads are magnetized on theright-hand side. This setup mimics the lateral mixing that was createdby the original benchtop HERMES device and combines it with inertialmixing that is enabled by gravity. One advantage of this embodiment isthat this protocol was designed to require minimal training: standardprotocol for plasma collection requires the inversion of the collectiontube for 5-7 times to ensure that the anticoagulant is mixed evenly. TheHERMES sleeve takes advantage of this requirement to perform theeffective mixing of magnetic beads and aggregation agent.

EXAMPLES

The following examples are intended to illustrate particular embodimentsof the present invention, but are by no means intended to limit thescope of the present invention.

Example 1 H.E.R.M.E.S: Rapid Blood-Plasma Separation at thePoint-of-Need

Abstract

The global healthcare landscape is experiencing increasing demand forCLIA-waived testing facilities that offer diagnostic capabilities atlower costs and greater convenience than traditional laboratory testing.While several new diagnostic tools have emerged to fulfill testingrequirements in these environments, centrifuges have been stymied fromtransitioning to the point-of-need as the US Food and DrugAdministration (FDA) classifies them as mostly unsuitable for use inCLIA-waived environments. Limitations in sample processing capabilitiesadversely affects the ability for CLIA-waived testing environments tooffer a broad testing portfolio and present-day diagnostics arebottlenecked by the requirement for centrifugation. Here we present theHigh Efficiency Rapid Magnetic Erythrocyte Separator (H.E.R.M.E.S), arapid low-cost technology that can perform the separation of red bloodcells from plasma at a fraction of the time and cost of that of acentrifuge. We demonstrate that H.E.R.M.E.S is able to obtainhighly-pure plasma (greater than 99.9% purity) at less than 2 minutesper test. Further, we detail that it is an easy-to-use method capable ofbeing incorporated with present-day diagnostic technologies and provethat it is superior to existing alternatives to centrifugation byvalidation with a ferritin lateral flow test. H.E.R.M.E.S is a suitablealternative for centrifugation in point-of-need settings and aims tofacilitate the decentralization of commercial blood testing.

H.E.R.M.E.S Device Design Landscape:

H.E.R.M.E.S has been designed with a specific intention of beingeasy-to-use. The process involves three main phases: capture, separationand extraction. In order to obtain separated plasma, the user need onlyfollow three steps (as outlined in FIG. 4A), i) collect the sample(typically with a finger-stick) and load the sample in a test tubeprecoated with functionalized magnetic beads, ii) place sample inH.E.R.M.E.S and wait for 2 minutes, iii) remove sample and extractplasma using a capillary tube. Once the sample has been extracted, thesample can be used for further analysis or stored for future use.

The portable benchtop device itself consists simple onboard electronicsto enable automation (FIG. 4D). H.E.R.M.E.S employs a small linearsolenoid that actuates a magnetic field in a specific direction withrespect to the sample to create a mixing effect. This ensures that thebeads are able to capture the erythrocytes in the sample. The deviceoccupies a small footprint and can be powered by any standard electricaloutlet. Once the device is plugged in, the actuation startsautomatically and proceeds for 90 seconds. After 90 seconds, thesolenoid turns off and the magnetic beads are concentrated by the magneton one side of the sample holder. The user then employs a smallcapillary tube to uptake the separated plasma. While the currentiteration requires the use of an outlet, it can be easily re-engineeredto include a portable battery pack instead. H.E.R.M.E.S was specificallydesigned to enable sample processing in point-of-need settings and wasdesigned for semi-autonomous operation to minimize the need for manualintervention by the user.

Magnetic Bead Capture of Erythrocytes:

Magnetic bead based capture has been adopted several biologicalapplications such as DNA extraction, peptidome assessment andimmunocapture (22-24). The technique is particularly useful forcapturing a small amount of analyte as the beads can be concentrated toyield a higher limit of detection (25). H.E.R.M.E.S tackles the oppositeproblem: a single microliter of human blood can contain up to 6 millionblood cells. This resulted in a significant challenge as it wasnecessary to capture all the erythrocytes in the sample without the needfor dilution.

At first glance, it would appear a simple assessment of the bindingcapacity of the magnetic beads would be sufficient in order determinethe minimum number of beads required to capture all the blood cells inthe sample. A “brute force” approach as such would be appropriate forseparation but would suffer from a lack of scalability. Further, thisapproach would have a fragile dependence on the sample size with acost-scaling directly proportional to the number of cells that wouldneed to be captured. In order to maintain a cost-effective scalingprinciple, H.E.R.M.E.S uses an aggregation agent that groups red bloodcells together during the capture phase, thereby reducing the effectivenumber of cells that need to be captured (See FIG. 2). By aggregatingthe cells prior to the capture, we are able to demonstrate anapproximate 2000-fold increase in binding capacity of the beads usingthis aggregation agent. H.E.R.M.E.S is unique in comparison to previousworks in literature that employ aggregation enhanced capture as it isperformed in an easily accessible format that does not requirespecialized filtration paper or microfluidic setup (26,27). Ourestimations reveal that on average, one bead is able to capture up to100 cells due to the aggregation effect. Approximately 5.4 mg of bloodcells are captured with close to a tenth of the amount of beads.

Evaluation of H.E.R.M.E.S Using Human Samples

We used H.E.R.M.E.S to process blood samples from 15 individuals andanalyzed the plasma obtained after extraction (Table 1). We demonstratean average purity greater than 99.9% (less than 20 cells/μL counted) andan average extraction time of 108 seconds to obtain 90% of the availableplasma. H.E.R.M.E.S demonstrates a high efficiency to capture andseparate red blood cells irrespective of blood type and hematocritlevels. To further demonstrate the ability of aggregation to enhance thecapture rate, we used H.E.R.M.E.S to separate erythrocytes inartificially spiked blood samples that have abnormally high hematocrit(FIG. 6). We hypothesize that the aggregation effect scales non-linearlywith an increase in the number of red blood cells. The highconcentration of blood cells decreases the interaction space leading toeffective binding in these samples. H.E.R.M.E.S can perform reliablywith an increase in red blood cells and is able to obtain highly pureplasma regardless of the number of blood cells. Factoring in the cost ofthe aggregation agent and the beads, we expect H.E.R.M.E.S to cost lessthan $2 per separation test.

TABLE 1 Average Purity of Obtained Plasma 99.95 ± 0.05% Average Time forExtraction   108 ± 21 seconds Average Volume Obtained  17.2 ± 1.96 μL

As shown in Table 1, data collected from testing 15 human blood sampleswith varying ages, hematocrits and blood types. A starting volume of 40μL was used for each test. Samples were run in duplicate and thestandard deviation is reported. Average volume of uncontaminated plasmaobtained from centrifugation was 18.2 μL. All samples were purifiedusing 0.625 mg of magnetic beads and 200 μg of aggregation agent.

Quantifying the Effect of Aggregation on Performance

Aggregation reduces the number of effective targets that need to becaptured to obtain highly pure plasma. To understand the dependence ofthe aggregation capabilities on separation performance, we tested thecell capture rate with varying levels of aggregation agent. The cellcapture rate was indirectly inferred by calculating the purity of theplasma obtained after separation. We then compared the purity of plasmaobtained using different sample volumes to assess the scalability of thetechnique. As seen in FIG. 7, it can be noted that a concentration ofabout 3.5 ug/mL of aggregation agent is sufficient to obtain plasma ofhigh purity (greater than 99%). We also observe a non-linearrelationship wherein the binding capacity is amplified several-fold asthe amount of agent is increased. We expect the curve to be pushedfurther out in the regime where the concentration of the aggregationagent is low. However, there exists a critical concentration thatcreates the level of aggregation necessary to obtain highly pure plasma(greater than 99%). In contrast, we also studied the effect ofincreasing the number of magnetic beads on performance and noted thatthe amount of beads does not significantly affect the separation ofperformance.

Integration with Existing Diagnostic Testing Platforms

We demonstrate the ability of H.E.R.M.E.S to augment the performance ofpresent day diagnostics by incorporating the technology with lateralflow test strips—a common diagnostic platform often implemented for useat the point-of-need. Ferritin lateral flow strips previously describedby Srinivasan et al. (28) were tested using 10 human blood samples. Wecompared the performance of H.E.R.M.E.S against commercially availablefiltration paper (MDI). A calibration curve was built with a lineartrendline fit to compare the performance of the two test cases (FIG. 8).In general, we observed that H.E.R.M.E.S leveraged higher quantitativepower from the regression model used in each of these cases. ROC curveswere plotted using the Delong method (29) and a difference inperformance was established. Further, as seen in FIG. 8 a higher slope(46%) was noted for test strips that incorporated plasma obtained fromH.E.R.M.E.S indicating greater quantitative ability across theconcentration range of interest. More importantly, H.E.R.M.E.S was ableto demonstrate a significantly reduced average coefficient of variation(6% vs 21%) further proving that it is capable of advancing theperformance of point-of-need testing platforms.

Discussion and Conclusion

H.E.R.M.E.S leverages the elementary concept of magnetic separation tocarry out a challenging process of separating unwanted cellular materialfrom a sample. The method was specifically designed to be low-cost,rapid and minimally complex. The system was designed to allow formaximal automation and minimal intervention from the user whilemaintaining a low-cost. These characteristics make H.E.R.M.E.S apromising alternative to the traditional lab centrifuge in settingswhere a centrifuge cannot operate, whether due to regulatory constraintsor resource limitations. Further, while we validated H.E.R.M.E.S with acommonly available point-of-need testing platform (lateral flow tests),it is universal and can be used to simplify purification for virtuallyany diagnostic test that requires plasma or serum as an input. Whilethere are a handful of sophisticated commercial platforms that arecapable of performing immunoassay chemistry without the need forseparating red blood cells, H.E.R.M.E.S will enable the use of standarddiagnostic techniques in low-resource settings where other alternativesare not feasible. Further, the high recovery rate (>90% of availableserum), the low time for recovery, the low cost and ease of use of thesystem make it superior to solutions that have been demonstrated inliterature (5, 8, 30).

H.E.R.M.E.S has the potential to impact the blood testing industry dueto its ability to offer the separation efficiency of a centrifuge at afraction of the time and cost. H.E.R.M.E.S seeks to facilitate theimplementation of present-day diagnostic tools at the point-of-need byintegrating into CLIA-waived testing environments where centrifuges arecurrently unable to operate. H.E.R.M.E.S can enable rapid front-endsample processing to help prevent loss of sample quality in theseenvironments by ensuring that all red blood cells are removed prior toclinical chemistry testing. We envision H.E.R.M.E.S having immediateapplicability in advancing molecular diagnostics such as PCR to thepoint-of-need as it can integrate easily in to existing methods fortranslational PCR. Particularly, the isolation of white blood cellsmakes an interesting use case for infectious disease detection (31, 32)and genetic sequencing (33). In addition to being highly scalable due tothe low cost of raw materials that are involved in fabrication,H.E.R.M.E.S is able to perform highly efficient blood plasma separationwithin 2 minutes at less than $2 per test and is suitable for use inresource-limited settings. While the current iteration of H.E.R.M.E.S isonly capable of accommodating one sample, we envision that a futureprototype will possess parallel sample processing capabilities as theunderlying technique is highly scalable. Further, the stand-alone systemis easy to use and can be adopted by users irrespective of their priormedical training making the H.E.R.M.E.S a unique method to performblood-plasma separation at the point-of-need.

Materials and Methods

Preparing Magnetic beads: Magnetic beads (ProMag HP, Bangs Labs)suspended in a 50 mM IVIES Buffer were conjugated to a Anti Red BloodCell antibody (Rockland Antibodies) by incubating the sample in anend-over-end mixer for 12-15 hours. After conjugation, the supernatantwas removed and replaced with a storage buffer (10 mM Tris Buffer, pH 8,0.05% Bovine Serum Albumin, 0.05% Proclin 300). The beads were stored at4-8° C. in liquid form and are stable up to several months. Prior totesting, 325 μg of beads and 200 μg of antibody were loaded in a singlePCR tube (Eppendorf) and dried in a vacuum centrifuge (EppendorfVacufuge 5301) for 30 minutes. The antibody is added separately toinduce clumping of the red blood cells to reduce the number of effectivetargets during the capture process. Dried beads were then used for testsor stored at 4-8° C. The dried beads demonstrated a shelf life of up tothree months when sealed in a dark container.

H.E.R.M.E.S benchtop unit: A small portable benchtop unit was designedin Sledworks and printed using a Objet 3D printer. The device itselfconsists of a microcontroller (Teensy 3.2, Sparkfun), a few transistorsand a 12V actuating solenoid (Adafruit Industries). Two circularneodymium magnets (K&J Magnetics) were mounted on a 3D printed holderand attached to the solenoid. The device also has a power input jackthat can be connected to standard 12V, 0.5 A wall power supply.

Analysis of Plasma: Upon separation of plasma from the red blood cellsusing H.E.R.M.E.S, a capillary tube (Microcaps, Drummond Scientific) wasused to extract the serum from the sample and transferred into anothertest tube. Once transferred, the serum was diluted 5 times and mixed ina 1:1 ratio with trypan blue stain. Once stained, the serum was loadedinto a disposable hemocytometer (C-Chip, Cyto Diagnostics) and cellcounting was performed under a bright field microscope. The number ofcells counted were then used to estimate the purity of the plasmaobtained.

High Hematocrit Samples: Blood samples with abnormal hematocrits wereprepared with type 0 human red blood cells suspended in alsever'ssolution (Innovative Research). The blood cells were spun down andconcentrated in a centrifuge and resuspended to abnormally highhematocrit values (70, 80 and 90%)

Lateral Flow Test: Ferritin strips were manufactured similar to workmentioned in Srinivasan et al. We prepared two batches of test stripswith blood filtration membranes (Type FR-1 (0.35) MDI membranetechnologies) used as a sample pad. The FR-1 is a passive forwardflowing filtration membrane that has a thickness of 0.35 mm and capacityof 30 μL/cm². 10 human blood samples (Innovative Research) were thenused for testing. For the strips that used the filtration membranes, a3-minute incubation period was added at the beginning of the test toallow the plasma to filter through the membrane. This was followed bythe application of 40 uL of running buffer to start the test. For thetest cases that used plasma from H.E.R.M.E.S, the test was immediatelystarted by using a 15 μL the capillary tube to apply the plasma onto thesample pad followed by application of 40 μL of running buffer. We notethat these test strips could also have been used as a dipstick format,wherein the sample pad is dipped in the sample holder to start the test.After 30 minutes, the test strips were imaged using the TIDBIT(previously mentioned by Lu et al. (34)) and a calibration curve wasbuilt using custom python code. Actual ferritin values were obtainedusing a SIEMENS Immulite1000 immunoassay analyzer.

REFERENCES

Citation of a reference herein shall not be construed as an admissionthat such reference is prior art to the present invention. Allreferences cited herein are hereby incorporated by reference in theirentirety. Below is a listing of various references relating to thepresent disclosure:

-   1. Global Blood Testing Market Size & Share|Industry Report,    2018-2024 [Internet]. [cited 2018 Jun. 14]. Available from:    https://www.grandviewresearch.com/industry-analysis/blood-testing-market-   2. Al-Soud W A, Radstrom P. Purification and Characterization of    PCR-Inhibitory Components in Blood Cells. J Clin Microbiol. 2001    Feb. 1; 39(2):485-93.-   3. Mabey D, Peeling R W, Ustianowski A, Perkins M D. Diagnostics for    the developing world: Tropical infectious diseases. Nat Rev    Microbiol. 2004 March; 2(3):231-40.-   4. Recommendations for Clinical Laboratory Improvement Amendments of    1988 (CLIA) WaiverApplications for Manufacturers of In Vitro    Diagnostic Device—Guidance for Industry and Food and Drug    Adminstration Staff [Internet]. United States Food and Drug    Adminstration (FDA); 2008. Available from:    https://www.fda.gov/downloads/MedicalDevices/DeviceRegulationandGuidance/GuidanceDocuments/ucm070890.pdf-   5. Liu C, Mauk M, Gross R, Bushman F D, Edelstein P H, Collman R G,    et al. Membrane-Based, Sedimentation-Assisted Plasma Separator for    Point-of-Care Applications. Anal Chem. 2013 Nov. 5; 85(21):10463-70.-   6. Posthuma-Trumpie G A, Korf J, van Amerongen A. Lateral flow    (immuno)assay: its strengths, weaknesses, opportunities and threats.    A literature survey. Anal Bioanal Chem. 2009 January; 393(2):569-82.-   7. Xu Q, Xu H, Gu H, Li J, Wang Y, Wei M. Development of lateral    flow immunoassay system based on superparamagnetic nanobeads as    labels for rapid quantitative detection of cardiac troponin I. Mater    Sci Eng C. 2009 April; 29(3):702-7.-   8. Son J H, Lee S H, Hong S, Park S, Lee J, Dickey A M, et al.    Hemolysis-free blood plasma separation. Lab Chip. 2014;    14(13):2287-92.-   9. Mielczarek W S, Obaje E A, Bachmann T T, Kersaudy-Kerhoas M.    Microfluidic blood plasma separation for medical diagnostics: is it    worth it? Lab Chip. 2016; 16(18):3441-8.-   10. Kersaudy-Kerhoas M, Sollier E. Micro-scale blood plasma    separation: from acoustophoresis to egg-beaters. Lab Chip. 2013;    13(17):3323.-   11. Gossett D R, Weaver W M, Mach A J, Hur S C, Tse H T K, Lee W, et    al. Label-free cell separation and sorting in microfluidic systems.    Anal Bioanal Chem. 2010 August; 397(8):3249-67.-   12. Bhagat A A S, Bow H, Hou H W, Tan S J, Han J, Lim C T.    Microfluidics for cell separation. Med Biol Eng Comput. 2010    October; 48(10):999-1014.-   13. Chen X, Cui D, Liu C, Li H. Microfluidic chip for blood cell    separation and collection based on crossflow filtration. Sens    Actuators B Chem. 2008 Mar. 14; 130(1):216-21.-   14. Chin C D, Linder V, Sia S K. Commercialization of microfluidic    point-of-care diagnostic devices. Lab Chip. 2012; 12(12):2118.-   15. Brown J, Theis L, O'Connor K, Kerr L, Uthman M, Oden Z M, et al.    A Hand-Powered, Portable, Low-Cost Centrifuge for Diagnosing Anemia    in Low-Resource Settings. Am J Trop Med Hyg. 2011 Aug. 1;    85(2):327-32.-   16. Wong A P, Gupta M, Shevkoplyas S S, Whitesides G M. Egg beater    as centrifuge: isolating human blood plasma from whole blood in    resource-poor settings. Lab Chip. 2008; 8(12):2032.-   17. Bhamla M S, Benson B, Chai C, Katsikis G, Johri A, Prakash M.    Hand-powered ultralow-cost paper centrifuge. Nat Biomed Eng. 2017    Jan. 10; 1(1):0009.-   18. Mariella R. Sample preparation: the weak link in    microfluidics-based biodetection. Biomed Microdevices. 2008    December; 10(6):777-84.-   19. Street P. Requirements for high impact diagnostics in the    developing world.:8.-   20. Dineva M A, Mahilum-Tapay L, Lee H. Sample preparation: a    challenge in the development of point-of-care nucleic acid-based    assays for resource-limited settings. The Analyst. 2007;    132(12):1193.-   21. Plebani M. Does POCT reduce the risk of error in laboratory    testing? Clin Chim Acta. 2009 June; 404(1):59-64.-   22. Caldarelli-Stefano R, Vago L, Bonetto S, Nebuloni M, Costanzi G.    Use of magnetic beads for tissue DNA extraction and IS6110    Mycobacterium tuberculosis PCR. Mol Pathol. 1999 Jun. 1;    52(3):158-60.-   23. Fiedler G M, Baumann S, Leichtle A, Oltmann A, Kase J, Thiery J,    et al. Standardized Peptidome Profiling of Human Urine by Magnetic    Bead Separation and Matrix-Assisted Laser Desorption/Ionization    Time-of-Flight Mass Spectrometry. Clin Chem. 2007 Mar. 1;    53(3):421-8.-   24. Sista R S, Eckhardt A E, Srinivasan V, Pollack M G, Palanki S,    Pamula V K. Heterogeneous immunoassays using magnetic beads on a    digital microfluidic platform. Lab Chip. 2008; 8(12):2188.-   25. Tang D, Zhong Z, Niessner R, Knopp D. Multifunctional magnetic    bead-based electrochemical immunoassay for the detection of    aflatoxin B1 in food. The Analyst. 2009; 134(8):1554.-   26. Tripathi S, Prabhakar A, Kumar N, Singh S G, Agrawal A. Blood    plasma separation in elevated dimension T-shaped microchannel.    Biomed Microdevices. 2013 June; 15(3):415-25.-   27. Yang X, Forouzan O, Brown T P, Shevkoplyas S S. Integrated    separation of blood plasma from whole blood for microfluidic    paper-based analytical devices. Lab Chip. 2012; 12(2):274-80.-   28. Srinivasan B, O'Dell D, Finkelstein J L, Lee S, Erickson D,    Mehta S. iron Phone: Mobile device-coupled point-of-care diagnostics    for assessment of iron status by quantification of serum ferritin.    Biosens Bioelectron. 2018 January; 99:115-21.-   29. DeLong E R, DeLong D M, Clarke-Pearson D L. Comparing the areas    under two or more correlated receiver operating characteristic    curves: a nonparametric approach. Biometrics. 1988 September;    44(3):837-45.-   30. Yeh E-C, Fu C-C, Hu L, Thakur R, Feng J, Lee L P. Self-powered    integrated microfluidic point-of-care low-cost enabling (SIMPLE)    chip. Sci Adv. 2017 March; 3(3):e1501645.-   31. Whitby D, Boshoff C, Hatzioannou T, Weiss R., Schulz T., Howard    M., et al. Detection of Kaposi sarcoma associated herpesvirus in    peripheral blood of HIV-infected individuals and progression to    Kaposi's sarcoma. The Lancet. 1995 September; 346(8978):799-802.-   32. Wang W-K, Sung T-L, Tsai Y-C, Kao C-L, Chang S-M, King C-C.    Detection of Dengue Virus Replication in Peripheral Blood    Mononuclear Cells from Dengue Virus Type 2-Infected Patients by a    Reverse Transcription-Real-Time PCR Assay. J Clin Microbiol. 2002    Dec. 1; 40(12):4472-8.-   33. Sigurdson A J, Hauptmann M, Chatterjee N, Alexander B H, Doody M    M, Rutter J L, et al. Kin-cohort estimates for familial breast    cancer risk in relation to variants in DNA base excision repair,    BRCA1 interacting and growth factor genes. BMC Cancer [Internet].    2004 December [cited 2018 Sep. 5]; 4(1). Available from:    http://bmccancer.biomedcentral.com/articles/10.1186/1471-2407-4-9-   34. Lu Z, O'Dell D, Srinivasan B, Rey E, Wang R, Vemulapati S, et    al. Rapid diagnostic testing platform for iron and vitamin A    deficiency. Proc Natl Acad Sci. 2017 Dec. 19; 114(51):13513-8.

First Set of Examples of Embodiments of the Present Disclosure

A1. A device for use in magnetic separation of a target biologicalentity from a fluid sample, said device comprising:

a magnetic separation chamber configured to receive a fluid sample formagnetic separation, wherein said magnetic separation chamber comprisestwo magnets mounted on opposing sidewalls a sufficient distance from oneanother so as to prevent interference between the magnetic field of eachmagnet, and wherein the magnetic separation chamber is configured toreceive and maintain the fluid sample at a position between the twomagnets during operation of the device; and

an actuator functionally coupled to the magnetic separation chamber,wherein said actuator comprises a linear actuator configured to move themagnetic separation chamber laterally in a side-to-side motion so as tokeep the two magnets in line with the fluid sample during operation ofthe actuator.

A2. The device according to A1, wherein the magnetic separation chamberis configured to receive a fluid sample having a volume of up to 200microliters.

A3. The device according to A1, wherein the magnetic separation chamberis configured to receive a vessel comprising a tube or other suitablecontainer containing the fluid sample.

A4. The device according to A1, wherein the magnets are circularmagnets.

A5. The device according to A1, wherein the magnets are neodymiummagnets.

A6. The device according to A1, wherein the actuator is an actuatingsolenoid [linear actuator solenoid].

A7. The device according to A6, wherein the actuating solenoid is a 12volt actuating solenoid.

A8. The device according to A1 further comprising a housing unitconfigured to house the magnetic separation chamber and the actuator.

A9. The device according to A8, wherein the housing unit comprises abase portion and a top cover portion, wherein the base portion isconfigured to hold the magnetic separation chamber and the actuator, andwherein the top cover portion is configured to fit over and cover thebase portion and its contents.

A10. The device according to A9, wherein the top cover portion furthercomprises an opening for inserting the fluid sample into a positionbetween the two magnets of the magnetic separation chamber.

A11. The device according to A8, wherein the housing unit is configuredto further house onboard electronics components effective to operate theactuator and to enable automation of the device.

A12. The device according to A11, wherein the onboard electronicscomponents comprise at least one component selected from the groupconsisting of a microcontroller, a transistor, and a power input jack.

A13. The device according to A12, wherein the power input jack issuitable for use with a standard 12 volt, 0.5 ampere wall power supply.

A14. The device according to A1, wherein the device is in a form of aportable benchtop device.

A15. The device according to A14, wherein the portable benchtop devicehas dimensions not greater than 4 centimeters in height, 5 centimetersin width, and 8 centimeters in length.

A16. The device according to A1, wherein the device is in a formsuitable for use for point-of-need diagnostics of the fluid sample.

B1. A system for use in magnetic separation of a target biologicalentity from a fluid sample, said system comprising:

at least one device according to any one of A1-A16;

a plurality of magnetic beads each functionalized to bind to the targetbiological entity, thereby being effective to capture and separate thetarget biological entity from the fluid sample.

B2. The system according to B1 further comprising an aggregation agentconfigured to group together an aggregate of a plurality of the targetbiological entities prior to the capture and separation of the targetbiological entity from the fluid sample.

B3. The system according to B2, wherein said aggregation agent isselected from the group consisting of an antibody, a protein, dextran,polyvinylpyrollidone, polyoxyethylene, fibrinogen, immunoglobulin IgMand IgG, calcium chloride, and combinations thereof.

B4. The system according to B2, wherein the target biological entity arered blood cells and the aggregation agent is an antibody that binds tored blood cells non-specifically, irrespective of blood type.

B5. The system according to B2, wherein the aggregation agent is anantibody that binds to a surface marker of red blood cells.

B6. The system according to B5, wherein the surface marker of the redblood cell is selected from the group consisting of CD235a and TER119.

B7. The system according to B2, wherein the plurality of magnetic beadsand the aggregation agent are contained in a vessel comprising a tube orother suitable container for containing the fluid sample duringoperation of the device.

B8. The system according to B2, wherein the plurality of magnetic beadsand the aggregation agent are provided together in a storage buffer.

B9. The system according to B2, wherein the plurality of magnetic beadsare conjugated to the aggregation agent prior to adding the fluidsample.

B10. The system according to B2, wherein the plurality of magnetic beadsand the aggregation agent are dried directly in a collection containerprior to adding the fluid sample.

B11. The system according to B10, wherein the collection container is avessel comprising a tube or other suitable container for containing thefluid sample during operation of the device.

B12. The system according to B1, wherein the target biological entity isselected from the group consisting of white blood cells, contaminants,waste products, and excess reagents contained in the fluid sample.

B13. The system according to B1 further comprising a collectioncomponent for collecting a supernatant produced after capture andseparation of the target biological entity from the fluid sample.

B14. The system according to B13, wherein the collection component is acapillary tube configured to passively uptake the supernatant.

B15. The system according to B13, wherein the fluid sample is a bloodsample, the target biological entity comprises red blood cells, and thesupernatant comprises plasma.

C1. A method for separating a target biological entity from a fluidsample, said method comprising the steps of:

providing a device according to any one of A1-A16;

combining the fluid sample with a plurality of magnetic beadsfunctionalized to bind to the target biological entity, wherein thecombining is performed in a vessel comprising a tube or other suitablecontainer for containing the fluid sample during operation of thedevice;

positioning the vessel containing the fluid sample and thefunctionalized magnetic beads between the two magnets of the magneticseparation chamber of the device; and

operating the device in a manner sufficient to capture the targetbiological entity on the functionalized magnetic beads, thereby yieldinga supernatant free of or substantially free of said target biologicalentity.

C2. The method according to C1, wherein the operating step comprisesrunning the actuator to enable the magnetic separation chamber tofunction to capture the target biological entity with the functionalizedmagnetic beads and concentrate them proximate to just one of the twomagnets, thereby separating the target biological entity from the fluidsample.

C3. The method according to C2, wherein running the actuator comprisesmoving the magnetic separation chamber laterally in a side-to-sidemotion with respect to the fluid sample for a sufficient number ofstrokes to homogenously distribute the functionalized magnetic beadsamong the target biological entity and concentrate them proximate tojust one of the two magnets, thereby separating the target biologicalentity from the fluid sample.

C4. The method according to C3, wherein the number of strokes is notgreater than 100 strokes, not greater than 50 strokes, not greater than40 strokes, not greater than 30 strokes, not greater than 20 strokes, ornot greater than 10 strokes.

C5. The method according to C3, wherein the sufficient number of strokesis completed within not more than 180 seconds, not more than 150seconds, not more than 120 seconds, not more than 90 seconds, not morethan 60 seconds, or not more than 30 seconds.

C6. The method according to C1 further comprising collecting thesupernatant from the vessel.

C7. The method according to C6, wherein the collecting is performedusing a capillary tube.

C8. The method according to C1 further comprising collecting andanalyzing the supernatant and/or the captured target biological entityusing one or more diagnostic tool or technique of interest.

C9. The method according to C1, wherein the method does not involve acentrifugation step to separate the target biological entity from thefluid sample.

C10. The method according to C1, wherein the magnetic beads areconjugated to an aggregation agent prior to the combining step, andwherein said aggregation agent is configured to group together anaggregate of a plurality of the target biological entities prior to thecapture and separation of the target biological entity from the fluidsample.

C11. The method according to C10, wherein said aggregation agent isselected from the group consisting of an antibody, a protein, dextran,polyvinylpyrollidone, polyoxyethylene, fibrinogen, immunoglobulin IgMand IgG, calcium chloride, and combinations thereof.

C12. The method according to C10, wherein the target biological entityare red blood cells and the aggregation agent is an antibody that bindsto red blood cells non-specifically, irrespective of blood type.

C13. The method according to C10, wherein the aggregation agent is anantibody that binds to a surface marker of red blood cells.

C14. The method according to C13, wherein the surface marker of the redblood cell is selected from the group consisting of CD235a and TER119.

C15. The method according to C10, wherein the plurality of magneticbeads and the aggregation agent are contained in a vessel comprising atube or other suitable container for containing the fluid sample duringoperation of the device.

C16. The method according to C10, wherein the plurality of magneticbeads and the aggregation agent are provided together in a storagebuffer.

C17. The method according to C10, wherein the plurality of magneticbeads are conjugated to the aggregation agent prior to adding the fluidsample.

C18. The method according to C10, wherein the plurality of magneticbeads and the aggregation agent are dried directly in a collectioncontainer prior to adding the fluid sample.

C19. The method according to C18, wherein the collection container is avessel comprising a tube or other suitable container for containing thefluid sample during operation of the device.

C20. The method according to C1, wherein the target biological entity isselected from the group consisting of white blood cells, contaminants,waste products, and excess reagents contained in the fluid sample.

C21. The method according to C1 further comprising a collectioncomponent for collecting a supernatant produced after capture andseparation of the target biological entity from the fluid sample.

C22. The method according to C21, wherein the collection component is acapillary tube configured to passively uptake the supernatant.

C23. The method according to C21, wherein the fluid sample is a bloodsample, the target biological entity comprises red blood cells, and thesupernatant comprises plasma.

Second Set of Examples of Embodiments of the Present Disclosure SleeveDevice

A1. A device for use in magnetic separation of a target biologicalentity from a fluid sample, said device comprising:

a magnetic separation chamber configured to receive a fluid sample formagnetic separation, wherein said magnetic separation chamber comprisesat least two magnets mounted on the surface or in the wall of themagnetic separation chamber,

a force provider configured to move the magnetic separation chamber in aside-to-side motion to mix and/or magnetize the fluid sample.

A2. The device according to A1, wherein the force provider is anactuator or a movable hand.

A3. The device according to A1, wherein the force provider is anactuator and the two magnets are mounted on opposing sidewalls asufficient distance from one another so as to prevent interferencebetween the magnetic field of each magnet,

wherein the magnetic separation chamber is configured to receive andmaintain the fluid sample at a position between the two magnets duringoperation of the device, and

wherein the actuator is functionally coupled to the magnetic separationchamber, said actuator comprising a linear actuator configured to movethe magnetic separation chamber laterally in a side-to-side motion so asto keep the two magnets in line with the fluid sample during operationof the actuator.

A4. The device according to A1, wherein the at least two magnets arefixed or movable along the wall of the magnetic separation chamber.

A5. The device according to A1, wherein the magnetic separation chamberfurther comprises at least two channels along the sidewall of thechamber to allow the at least two magnets movable in order to mix andmagnetize the sample.

A6. The device according to A1, wherein the at least two channels arelinear channels, curved channels, symmetric channels, cylindricalshaped, or tube shaped.

A7. The device according to A1, wherein the force provider is a manualforce provider or an automatic force provider.

A8. The device according to A1, wherein the fluid sample furthercomprises at least an aggregation agent configured to group together anaggregate of a plurality of the target biological entities.

A9. The device according to A1, wherein the device further comprises acollection component for collecting a supernatant produced after captureand separation of the target biological entity from the fluid sample,and wherein the collection component is cap comprising capillary tube.

A10. The device according to A1, said magnetic separation chambercomprising a durable plastic material or any other non-magneticmaterial.

A11. The device according to A1, wherein the at least two magnets areball magnets.

A12. The device according to A11, wherein the ball magnets are 3/16 inchball magnets.

A13. The device according to A11, wherein the magnetic separationchamber is in a form of a sleeve and comprises a substantially centralchannel for loading a vessel containing the fluid sample therein.

A14. The device according to A13, wherein the substantially centralchannel has a circumference suitable for a standard blood collectiontube.

A15. The device according to A14, wherein the substantially centralchannel has a circumference of approximately 10 mm and a depth ofapproximately 70-75 mm.

A16. The device according to A13, wherein the sleeve is configured tocontain a vessel comprising a 3 mL tube.

A17. The device according to A13, wherein the vessel can contain avolume of fluid sample selected from the group consisting of up to 200uL, up to 500 uL, up to 1 mL, up to 1.5 mL, up to 2.0 mL, up to 2.5 mL,up to 3.0 mL, and up to 5.0 mL.

A18. The device according to A13, wherein the vessels can contain avolume of fluid sample of 200 uL or greater.

A19. The device according to A13, wherein the sleeve is made with 3Dprinted plastic or any durable plastic or non-magnetic material.

A20. The device according to A13, wherein the sleeve is made withinjection molding techniques.

A21. The device according to A13, wherein the sleeve comprisesdimensions approximating those of a 10 mL blood tube, said dimensionscomprising approximately 16 mm in circumference and 100 mm in length forthe depth of the substantially central channel, wherein total size ofthe sleeve can alternatively scale up or down according to thesedimensions accordingly.

A22. The device according to A21, wherein the size of the sleeve can bescaled up at least by approximately 1.5 times.

B1 A method for separating a target biological entity from a fluidsample, said method comprising the steps of:

providing a device according to any one of A1-A22;

combining the fluid sample with a plurality of magnetic beadsfunctionalized to bind to the target biological entity, wherein thecombining is performed in a vessel comprising a tube or other suitablecontainer for containing the fluid sample during operation of thedevice;

positioning the vessel containing the fluid sample and thefunctionalized magnetic beads between the two magnets of the magneticseparation chamber of the device; and

operating the device in a manner sufficient to capture the targetbiological entity on the functionalized magnetic beads, thereby yieldinga supernatant free of or substantially free of said target biologicalentity.

B2. The method according to B1 further comprising collecting thesupernatant from the vessel.

B3. The method according to B1 further comprising collecting andanalyzing the supernatant and/or the captured target biological entityusing one or more diagnostic tool or technique of interest.

B4. The method according to B1, wherein the method does not involve acentrifugation step to separate the target biological entity from thefluid sample.

B5. The method according to B1, wherein the magnetic beads areconjugated to an aggregation agent prior to the combining step, andwherein said aggregation agent is configured to group together anaggregate of a plurality of the target biological entities prior to thecapture and separation of the target biological entity from the fluidsample.

B6. The method according to B5, wherein said aggregation agent isselected from the group consisting of an antibody, a protein, dextran,polyvinylpyrollidone, polyoxyethylene, fibrinogen, immunoglobulin IgMand IgG, calcium chloride, and combinations thereof.

B7. The method according to B5, wherein the target biological entity arered blood cells and the aggregation agent is an antibody that binds tored blood cells non-specifically, irrespective of blood type.

B8. The method according to B5, wherein the aggregation agent is anantibody that binds to a surface marker of red blood cells.

B9. The method according to B8, wherein the surface marker of the redblood cell is selected from the group consisting of CD235a and TER119.

B10. The method according to B5, wherein the plurality of magnetic beadsand the aggregation agent are contained in a vessel comprising a tube orother suitable container for containing the fluid sample duringoperation of the device.

B11. The method according to B5, wherein the plurality of magnetic beadsand the aggregation agent are provided together in a storage buffer.

B12. The method according to B5, wherein the plurality of magnetic beadsare conjugated to the aggregation agent prior to adding the fluidsample.

B13. The method according to B5, wherein the plurality of magnetic beadsand the aggregation agent are dried directly in a collection containerprior to adding the fluid sample.

B14. The method according to B13, wherein the collection container is avessel comprising a tube or other suitable container for containing thefluid sample during operation of the device.

B15. The method according to B1, wherein the target biological entity isselected from the group consisting of white blood cells, contaminants,waste products, and excess reagents contained in the fluid sample.

B16. The method according to B1 further comprising a collectioncomponent for collecting a supernatant produced after capture andseparation of the target biological entity from the fluid sample.

B17. The method according to B16, wherein the collection component is acapillary tube configured to passively uptake the supernatant.

B18. The method according to B16, wherein the fluid sample is a bloodsample, the target biological entity comprises red blood cells, and thesupernatant comprises plasma.

Illustrative embodiments of the processes, methods, and products of thepresent disclosure are described herein. It should be understood,however, that the description herein of the specific embodiments is notintended to limit the present disclosure to the particular formsdisclosed but, on the contrary, the intention is to cover allmodifications equivalents and alternatives falling within the spirit andscope of the invention by the appended claims. Thus, although thepresent invention has been described for the purpose of illustration, itis understood that such detail is solely for that purpose and variationscan be made by those skilled in the art without departing from thespirit and scope of the invention which is defined by the followingclaims.

What is claimed is:
 1. A device for use in magnetic separation of atarget biological entity from a fluid sample, said device comprising: amagnetic separation chamber configured to receive a fluid sample formagnetic separation, wherein said magnetic separation chamber comprisestwo magnets mounted on opposing sidewalls a sufficient distance from oneanother so as to prevent interference between the magnetic field of eachmagnet, and wherein the magnetic separation chamber is configured toreceive and maintain the fluid sample at a position between the twomagnets during operation of the device; and an actuator functionallycoupled to the magnetic separation chamber, wherein said actuatorcomprises a linear actuator configured to move the magnetic separationchamber laterally in a side-to-side motion so as to keep the two magnetsin line with the fluid sample during operation of the actuator.
 2. Thedevice according to claim 1, wherein the magnetic separation chamber isconfigured to receive a fluid sample having a volume of up to 200microliters.
 3. The device according to claim 1, wherein the magneticseparation chamber is configured to receive a vessel comprising a tubeor other suitable container containing the fluid sample.
 4. The deviceaccording to claim 1, wherein the magnets are circular magnets orneodymium magnets.
 5. The device according to claim 1 further comprisinga housing unit configured to house the magnetic separation chamber andthe actuator.
 6. The device according to claim 5, wherein the housingunit comprises a base portion and a top cover portion, wherein the baseportion is configured to hold the magnetic separation chamber and theactuator, and wherein the top cover portion is configured to fit overand cover the base portion and its contents.
 7. The device according toclaim 6, wherein the top cover portion further comprises an opening forinserting the fluid sample into a position between the two magnets ofthe magnetic separation chamber.
 8. The device according to claim 5,wherein the housing unit is configured to further house onboardelectronics components effective to operate the actuator and to enableautomation of the device.
 9. The device according to claim 8, whereinthe onboard electronics components comprise at least one componentselected from the group consisting of a microcontroller, a transistor,and a power input jack.
 10. The device according to claim 9, wherein thepower input jack is suitable for use with a standard 12 volt, 0.5 amperewall power supply.
 11. The device according to claim 1, wherein thedevice is in a form of a portable benchtop device.
 12. The deviceaccording to claim 11, wherein the portable benchtop device hasdimensions not greater than 4 centimeters in height, 5 centimeters inwidth, and 8 centimeters in length.
 13. The device according to claim 1,wherein the device is in a form suitable for use for point-of-needdiagnostics of the fluid sample.
 14. A system for use in magneticseparation of a target biological entity from a fluid sample, saidsystem comprising: at least one device according to claim 1; a pluralityof magnetic beads each functionalized to bind to the target biologicalentity, thereby being effective to capture and separate the targetbiological entity from the fluid sample.
 15. The system according toclaim 14 further comprising an aggregation agent configured to grouptogether an aggregate of a plurality of the target biological entitiesprior to the capture and separation of the target biological entity fromthe fluid sample.
 16. The system according to claim 15, wherein saidaggregation agent is selected from the group consisting of an antibody,a protein, dextran, polyvinylpyrollidone, polyoxyethylene, fibrinogen,immunoglobulin IgM and IgG, calcium chloride, and combinations thereof.17. The system according to claim 15, wherein the target biologicalentity are red blood cells and the aggregation agent is an antibody thatbinds to red blood cells non-specifically, irrespective of blood type.18. The system according to claim 15, wherein the aggregation agent isan antibody that binds to a surface marker of red blood cells.
 19. Thesystem according to claim 18, wherein the surface marker of the redblood cell is selected from the group consisting of CD235a and TER119.20. The system according to claim 15, wherein the plurality of magneticbeads and the aggregation agent are: (a) contained in a vesselcomprising a tube or other suitable container for containing the fluidsample during operation of the device; (b) provided together in astorage buffer; (c) conjugated to one another prior to adding the fluidsample; or (d) dried directly in a collection container prior to addingthe fluid sample.
 21. The system according to claim 20, wherein thecollection container is a vessel comprising a tube or other suitablecontainer for containing the fluid sample during operation of thedevice.
 22. The system according to claim 14, wherein the targetbiological entity is selected from the group consisting of white bloodcells, contaminants, waste products, and excess reagents contained inthe fluid sample.
 23. The system according to claim 14 furthercomprising a collection component for collecting a supernatant producedafter capture and separation of the target biological entity from thefluid sample.
 24. The system according to claim 23, wherein thecollection component is a capillary tube configured to passively uptakethe supernatant.
 25. The system according to claim 23, wherein the fluidsample is a blood sample, the target biological entity comprises redblood cells, and the supernatant comprises plasma.
 26. A method forseparating a target biological entity from a fluid sample, said methodcomprising the steps of: providing a device according to claim 1;combining the fluid sample with a plurality of magnetic beadsfunctionalized to bind to the target biological entity, wherein thecombining is performed in a vessel comprising a tube or other suitablecontainer for containing the fluid sample during operation of thedevice; positioning the vessel containing the fluid sample and thefunctionalized magnetic beads between the two magnets of the magneticseparation chamber of the device; and operating the device in a mannersufficient to capture the target biological entity on the functionalizedmagnetic beads, thereby yielding a supernatant free of or substantiallyfree of said target biological entity.